Background Accurate knowledge of the spinal structural functions is critical to understand the biomechanical factors that affect spinal pathology. Many studies have investigated the human vertebral motion both in vitro and in vivo.However, determination of in vivo motion of the vertebrae under physiologic loading conditions remains a challenge in biomedical engineering because of the limitations of current technology and the complicated anatomy of the spine.Methods For in vitro validation, a human lumbar specimen was imbedded with steel beads and moved to a known distance by an universal testing machine (UTM). The dual fluoroscopic system was used to capture the spine motion and reproduce the moving distance. For in vivo validation, a living subject moved the spine in various positions while bearing weight. The fluoroscopes were used to reproduce the in vivo spine positions 5 times. The standard deviations in translation and orientation of the five measurements were used to evaluate the repeatability of technique. The accuracy of vertebral outline matching with metallic marks matching technology was compared.Results The translation positions of the human lumbar specimen could be determined with a mean accuracy less than 0.35 mm and a mean repeatability 0.36 mm for the image matching technique. The repeatability of the method in reproducing in vivo human spine six degrees of freedom (6DOF) kinematics was less than 0.43 mm in translation and less than 0.65° in rotation. The accuracy of metallic marks and vertebral outline matching did not show significant difference.Conclusions Combining a dual fluoroscopic and computerized tomography imaging technique was accurate and reproduceable for noninvasive measurement of spine vertebral motion. The vertebral outline matching technique could be a useful technique for matching of vertebral positions and orientations which can evaluate and improve the efficacy of the various surgical treatments.